Technical Abstract:
MicroRNAs (miR) are small non-coding RNA transcripts (~22 nucleotides long) that have been shown to modulate diverse cell functions by interfering with translation and/or targeting mRNA for degradation. Since there is no information regarding microRNAs in bovine, we characterized this new class of RNA using tissues important for immune response and embryonic development. Alignment of human microRNA stem loop sequences (mir) against the bovine genome resulted in identification of 221 predicted bovine mirs. We validated some of these predictions by constructing and sequencing 3,209 cDNA clones made from small RNA fractions of bovine embryo, thymus, small intestine, abomasum, and mesenteric lymph nodes. This strategy resulted in identification of 129 putative mature microRNAs (miRs). Of these, 100 aligned to known human miRs, supporting their designation as bona fide miRs. An additional seven aligned to the complementary arm of a known human miR, and thus are novel miRs. Interestingly, 22 that do not have matches to human mirs displayed characteristic mir secondary structures, and 11 of these show phylogenetic conservation among other vertebrate species. Clustering of different tissues based on miR expression showed that similar tissues (abomasal and mesenteric lymph nodes) grouped together, whereas the most distinct tissue (embryonic) formed an out-group, with others (small intestine and thymus) placed in-between in the hierarchical tree. Clustering formed four major groups of miR expression, with each cluster having a characteristic signature of being selectively expressed in one or more tissue types. The miR26a was the most abundant miR, expressed in all the five tissues; whereas in human studies for the same tissues, it has been shown to be present in varying levels from high to low. This study revealed interesting insights into bovine miRNA expression such as extent of mir sequence conservation, prevalence of miR26a in all libraries and discovery of novel miRs.